VEGF Protein Levels Research Articles

MiR-20b targets AKT3 and modulates vascular endothelial growth factor-mediated changes in diabetic retinopathy.

Diabetic retinopathy (DR) is the leading cause of new-onset blindness. The roles of microRNAs in diabetic retinopathy are largely unknown. The aim of this study is to investigate the role of miR-20b in DR. Transfection of miR-20b mimic in high glucose (HG)-treated human retinal endothelial cells (HRECs) increased miR-20b expression and decreased the expression level of VEGF mRNA, while transfection of miR-20b inhibitor in control HRECs reduced the miR-20b expression with a corresponding increase of VEGF mRNA. In vitro functional assay showed that transfection of miR-20b mimic prevented HG-induced increase in transendothelial permeability and tube formation in HRECs. Transfection of miR-20b inhibitor or treatment of VEGF increased transendothelial permeability and tube formation in control HRECs. Luciferase reported assay showed that AKT3 is a target of miR-20b. Transfection of miR-20b mimic prevented the up-regulation of AKT3 induced by HG without changing the protein levels of other isoforms of AKT, and silencing of AKT3 caused decrease of VEGF mRNA and protein levels as well as prevented HG-induced increase in transendothelial permeability and tube formation. Finally, we showed that miR-20b was down-regulated in the retina and retinal endothelial cells in diabetic rats, with a correlated up-regulation of VEGF and AKT3. Intravitreal injection of miR-20b mimic in the diabetic rat significantly increased the miR-20b expression and decreased the expression levels of AKT3 and VEGF in the retina tissues, and intravitreal delivery of AKT3 siRNA in the diabetic rat significantly decreased the expressions of AKT3 and VEGF. Collectively, miR-20b is important for the regulation of VEGF-mediated changes in HRECs and rat retinal tissues under hyperglycemic conditions possibly via targeting AKT3.

β-cell insulin receptor deficiency during in utero development induces an islet compensatory overgrowth response.

The presence of insulin receptor (IR) on β-cells suggests that insulin has an autocrine/paracrine role in the regulation of β-cell function. It has previously been reported that the β-cell specific loss of IR (βIRKO) leads to the development of impaired glycemic regulation and β-cell death in mice. However, temporally controlled βIRKO induced during the distinct transitions of fetal pancreas development has yet to be investigated. We hypothesized that the presence of IR on β-cells during the 2nd transition phase of the fetal murine pancreas is required for maintaining normal islet development. We utilized a mouse insulin 1 promoter driven tamoxifen-inducible Cre-recombinase IR knockout (MIP-βIRKO) mouse model to investigate the loss of β-cell IR during pancreatic development at embryonic day (e) 13, a phase of endocrine proliferation and β-cell fate determination. Fetal pancreata examined at e19-20 showed significantly reduced IR levels in the β-cells of MIP-βIRKO mice. Morphologically, MIP-βIRKO pancreata exhibited significantly enlarged islet size with increased β-cell area and proliferation. MIP-βIRKO pancreata also displayed significantly increased Igf-2 protein level and Akt activity with a reduction in phospho-p53 when compared to control littermates. Islet vascular formation and Vegf-a protein level was significantly increased in MIP-βIRKO pancreata. Our results demonstrate a developmental role for the β-cell IR, whereby its loss leads to an islet compensatory overgrowth, and contributes further information towards elucidating the temporally sensitive signaling during β-cell commitment.

Paricalcitol Enhances the Chemopreventive Efficacy of 5-Fluorouracil on an Intermediate-Term Model of Azoxymethane-Induced Colorectal Tumors in Rats.

Colorectal cancer is a common cancer with high mortality rate. Despite being the standard anti-colorectal cancer drug, 5-fluorouracil (5-FU) exhibits only limited therapeutic benefits. Herein, we investigated whether paricalcitol, a synthetic vitamin D analogue with potential antitumor properties, would enhance the chemopreventive efficacy of 5-FU on an intermediate-term (15 weeks) model of colorectal tumors induced by azoxymethane (AOM) in rats. After AOM injection, 5-FU was administered during the 9th and 10th weeks (12 mg/kg/day for 4 days, then 6 mg/kg every other day for another 4 doses), whereas paricalcitol (2.5 μg/kg/day; 3 days/week) was given from the 7th to the 15th week. At week 15, the animals were euthanized and their resected colons were examined macroscopically and microscopically. Quantitative RT-PCR was used to measure the transcription activities of Wnt, β-catenin, DKK-1, CDNK-1A, NF-κB, and COX-2 genes, and ELISA was used to quantify the protein levels of β-catenin, COX-2, HSP90, and VEGF. IHC was additionally used to measure β-catenin, HSP90, and inducible nitric oxide synthase (iNOS). Compared with their individual therapy, combination of 5-FU and paricalcitol showed more significant reducing effect on numbers of grown tumors and large aberrant crypts foci. Mechanistically, paricalcitol and 5-FU had cooperated together to repress the expression of procancerous Wnt, β-catenin, NF-κB, COX-2, iNOS, VEGF, and HSP-90 more, and to upregulate the expression of antitumorigenesis DKK-1 and CDNK-1A, compared with their monotherapies. Our findings suggest that combined use of paricalcitol with 5-FU exhibits an augmenting chemopreventive effect against colorectal tumors, and might potentially be useful for chemoprevention in colorectal cancer patients. Cancer Prev Res; 9(6); 491-501. ©2016 AACR.

Uridine adenosine tetraphosphate acts as a proangiogenic factor in vitro through purinergic P2Y receptors.

Uridine adenosine tetraphosphate (Up4A), a dinucleotide, exerts vascular influence via purinergic receptors (PR). We investigated the effects of Up4A on angiogenesis and the putative PR involved. Tubule formation assay was performed in a three-dimensional system, in which human endothelial cells were cocultured with pericytes with various Up4A concentrations for 5 days. Expression of PR subtypes and angiogenic factors was assessed in human endothelial cells with and without P2Y6R antagonist. No difference in initial tubule formation was detected between Up4A stimulation and control conditions at day 2 In contrast, a significant increase in vascular density in response to Up4A was observed at day 5 Up4A at an optimal concentration of 5 μM promoted total tubule length, number of tubules, and number of junctions, all of which were inhibited by the P2Y6R antagonist MRS2578. Higher concentrations of Up4A (10 μM) had no effects on angiogenesis parameters. Up4A increased mRNA level of P2YRs (P2Y2R, P2Y4R, and P2Y6R) but not P2XR (P2X4R and P2X7R) or P1R (A2AR and A2BR), while Up4A upregulated VEGFA and ANGPT1, but not VEGFR2, ANGPT2, Tie1, and Tie2. In addition, Up4A increased VEGFA protein levels. Transcriptional upregulation of P2YRs by Up4A was inhibited by MRS2578. In conclusion, Up4A is functionally capable of promoting tubule formation in an in vitro coculture system, which is likely mediated by pyrimidine-favored P2YRs but not P2XRs or P1Rs, and involves upregulation of angiogenic factors.

Ranibizumab interacts with the VEGF-A/VEGFR-2 signaling pathway in human RPE cells at different levels

Vascular endothelial growth factor (VEGF) secreted by the retinal pigment epithelium (RPE) plays an important role in ocular homeostasis, but also in diseases, most notably age-related macular degeneration (AMD). To date, anti-VEGF drugs like ranibizumab have been shown to be most effective in treating these pathologic conditions. However, clinical trials suggest that the RPE could degenerate and perish through anti-VEGF treatment. Herein, we evaluated possible pathways and outcomes of the interaction between ranibizumab and human RPE cells (ARPE-19). Results indicate that ranibizumab affects the VEGF-A metabolism in RPE cells from an extra- as well as intracellular site. The drug is taken up into the cells, with the VEGF receptor 2 (VEGFR-2) being involved, and decreases VEGF-A protein levels within the cells as well as extracellularly. Oxidative stress plays a key role in various inflammatory disorders of the eye. Our results suggest that oxidative stress inhibits RPE cell proliferation. This anti-proliferative effect on RPE cells is significantly enhanced through ranibizumab, which does not inhibit RPE cell proliferation substantially in absence of relevant oxidative stress. Therefore, we emphasize that anti-VEGF treatment should be selected carefully in AMD patients with preexistent extensive RPE atrophy.

Role of thymosin beta 4 in hair growth.

Although thymosin beta 4 (Tβ4) is known to play a role in hair growth, its mechanism of action is unclear. We examined the levels of key genes in a Tβ4 epidermal-specific over-expressing mouse model and Tβ4 global knockout mouse model to explore how Tβ4 affects hair growth. By depilation and histological examination of the skin, we confirmed the effect of Tβ4 on hair growth, the number of hair shafts and hair follicle (HF) structure. The mRNA and protein expression of several genes involved in hair growth were detected by real-time PCR and western blotting, respectively. Changes in the expression of β-catenin and Lef-1, the two key molecules in the Wnt signaling pathway, were similar to the changes observed in Tβ4 expression. We also found that compared to the control mice, the mRNA and protein expression of MMP-2 and VEGF were increased in the Tβ4 over-expressing mice, while the level of E-cadherin (E-cad) remained the same. Further, in the Tβ4 global knockout mice, the mRNA and protein levels of MMP-2 and VEGF decreased dramatically and the level of E-cad was stable. Based on the above results, we believe that Tβ4 may regulate the levels of VEGF and MMP-2 via the Wnt/β-catenin/Lef-1 signaling pathway to influence the growth of blood vessels around HFs and to activate cell migration. Tβ4 may have potential for the treatment of hair growth problems in adults, and its effects should be further confirmed in future studies.

Effects of crocin and voluntary exercise, alone or combined, on heart VEGF-A and HOMA-IR of HFD/STZ induced type 2 diabetic rats.

Hyperglycemia is the main risk factor for microvascular complications in type 2 diabetes. Crocin and voluntary exercise have anti-hyperglycemic effects in diabetes. In this research, we evaluated the effects of crocin and voluntary exercise alone or combined on glycemia control and heart level of VEGF-A. Animals were divided into eight groups as: control (con), diabetes (Dia), crocin (Cro), voluntary exercise (Exe), crocin and voluntary exercise (Cro-Exe), diabetic-crocin (Dia-Cro), diabetic-voluntary exercise (Dia-Exe), diabetic-crocin-voluntary exercise (Dia-Cro-Exe). Type 2 diabetes was induced by a high-fat diet (4weeks) and injection of streptozotocin (STZ) (i.p, 35mg/kg). Animals received oral administration of crocin (50mg/kg) or performed voluntary exercise alone or together for 8weeks. Oral glucose tolerance test (OGTT) was performed on overnight fasted control, diabetic and treated rats after 8weeks of treatment. Then, serum insulin and heart VEGF-A protein levels were measured. Crocin combined with voluntary exercise significantly decreased blood glucose levels (p<0.001) and insulin resistance (HOMA-IR) (p<0.001) compared to diabetic group. VEGF-A level was significantly (p<0.01) lower in Dia group compared to control group. The combination of crocin and voluntary exercise significantly enhanced VEGF-A protein levels in Dia-Cro-Exe and Cro-Exe group compared to diabetic and control groups, respectively; p<0.001 and p<0.05. Crocin combined with voluntary exercise improved insulin resistance (HOMA-IR) and reduced glucose levels in diabetic rats. Since both crocin and voluntary exercise can increase VEGF-A protein expression in heart tissue, they probably are able to increase angiogenesis in diabetic animals.

The Influence Of Obesity‐Associated Metabolic Disturbance On The Profile Of Angiogenic Regulators And Capillary Number Within Skeletal Muscle

Skeletal muscle capillaries are critical determinants of nutrient delivery and utilization and capillary network density is thought to correlate with insulin sensitivity. Conflicting data exist regarding the consequence of obesity‐associated metabolic disturbances on skeletal muscle capillarization, with positive, negative and no effect having been reported. The purpose of this investigation is to assess the influence of obesity induced by high fat feeding or by leptin receptor mutation, on the angiogenic profile within the skeletal muscle microenvironment, and ultimately, on muscle capillarization. In FVB/n mice fed a high‐fat (HF) or normal chow (NC) diet for 16‐weeks, mRNA levels of VEGF‐A did not change (NC: 1.06 vs. HF: 1.13; P&gt;0.05) but VEGF protein levels were elevated (NC: 81 vs HF: 104 pg/mg; P =0.02). Relative FoxO1 protein levels increased in capillaries isolated from muscle of HF vs. NC mice (NC: 0.67 vs HF: 1.55; P =0.03). Capillary‐to‐fibre ratio (C:F) was not altered in HF‐ compared to NC‐ fed mice (2.22 and 2.04, P=0.34). In contrast, 13 week old mice with leptin receptor activity deficiency (Leprdb) had a significant reduction in VEGF‐A mRNA (1.85 vs. 1.09, P=0.03) and protein (WT: 98 vs Leprdb: 86 pg/mg; P&lt;0.05) relative to age‐matched C57BL/6 (WT) mice. Both FoxO1 (0.58 vs. 0.86) and THBS1 (0.37 vs. 0.81) mRNA levels tended to increase in Leprdb vs. WT mice. Skeletal muscle C:F was substantially reduced in Leprdb vs. WT mice (1.92 vs. 1.39, P&lt;0.001). Comprehensive profiling of angiogenic regulators will be conducted by microarray analysis. The skeletal muscle microenvironment differs substantially between HF and Leprdb models. Our data suggest that muscle capillarization is maintained during HF feeding due to a balance between pro and anti‐angiogenic factors, which may include VEGF‐A and FoxO1. This balance is disrupted in leptin receptor mutant mice, which results in a loss of capillaries and raises the intriguing possibility that leptin signaling itself modulates muscle capillarization. Thus, the Leprdb model may not be a suitable model to study obesity‐induced vascular alterations in skeletal muscle. Funded by CIHR.

Quercetin inhibits angiogenesis by targeting calcineurin in the xenograft model of human breast cancer

Vascular endothelial growth factor receptor 2 (VEGFR2) mediated calcineurin/nuclear factor of activated T-cells (NFAT) pathway is crucial in the angiogenesis of human breast cancer. Quercetin (Qu), a flavonoid known to possess anti-angiogenesis and antitumor properties, inhibited calcineurin activity in vitro. Herein, we performed a study in vivo to evaluate the effects of Qu on the angiogenesis in breast cancer. Female BALB/c nude mice were injected with MCF-7 cells into the mammary fat and were randomly divided into four groups. The animals were treated with vehicle solution, tamoxifen (TAM, 5.6mg/kg), tacrolimus (FK506, 3mg/kg), or Qu (34mg/kg) for 21 days, respectively. The results showed that, similar to TAM and FK506, Qu decreased tumor growth, limited oncocyte proliferation and promoted tumor necrosis. Anti-angiogenic actions of Qu were demonstrated as decreased serum VEGF (P<0.01), and sparse microvessel density (P<0.05). Qu significantly inhibited tumor calcineurin activities, and the inhibitory rate was 62.73% in Qu treated animals, compared to that was 72.90% in FK506 group (P>0.05). Effects of Qu on calcineurin/NFAT pathway were confirmed as decreased subcellular located levels of VEGF (P<0.05), VEGFR2 (P<0.05) and NFATc3 (P<0.01), downregulated gene expression of VEGF (P<0.05), VEGFR2 (P<0.05) and NFATc3 (P<0.01), reduced protein levels of VEGF (P<0.05), VEGFR2 (P<0.05), and NFATc3 (P<0.01) in tumor tissues. These findings indicate that Qu inhibit angiogenesis of human breast cancer xenograft in nude mice, which was associated with suppressing calcineurin activity and its regulated pathway activation.

The Vascular Endothelial Growth Factor Inhibitors Ranibizumab and Aflibercept Markedly Increase Expression of Atherosclerosis-Associated Inflammatory Mediators on Vascular Endothelial Cells.

IntroductionRecent studies have suggested that the VEGF inhibitors, Ranibizumab and Aflibercept may be associated with an excess of cardiovascular events, potentially driven by increasing atheroma instability, leading to plaque rupture and clinical events. Inflammation plays a key role in the progression of atherosclerotic plaque and particularly conversion to an unstable phenotype. Here, we sought to assess the in vitro effects of these drugs on the expression of key inflammatory mediators on endothelial cells.MethodsHuman coronary artery endothelial cells were co-incubated for 16h with Ranibizumab (0.11nM) or Aflibercept (0.45nM), as determined by each drug’s peak serum concentration (Cmax). Expression at protein (ELISA) and gene (RT-PCR) level of inflammatory chemokines CCL2, CCL5 and CXC3L1 as well as gene expression for the cell adhesion molecules VCAM-1, ICAM-1 and the key NF-κb protein p65 was assessed. VEGF-A protein levels were also determined.ResultsBoth drugs significantly increased chemokine, cell adhesion molecule (CAM) and p65 expression, while decreasing VEGF-A protein secretion. At equivalent Cmax concentrations, Aflibercept was significantly more pro-inflammatory than Ranibizumab. Reduction of secreted VEGF-A levels significantly attenuated inflammatory effects of both drugs, whereas blockade of the VEGF-A receptor or silencing of VEGF-A gene synthesis alone had no effect, suggesting that binding of drug to secreted VEGF-A is crucial in promoting inflammation. Finally, blockade of Toll-like receptor 4 significantly reduced inflammatory effects of both drugs.ConclusionWe demonstrated here, for the first time, that both drugs have potent pro-inflammatory effects, mediated via activation of Toll-like receptor 4 on the endothelial cell surface by drug bound to VEGF-A. Further studies are required to investigate whether these effects are also seen in vivo.

A small interfering RNA targeting vascular endothelial growth factor efficiently inhibits growth of VX2 cells and VX2 tumor model of hepatocellular carcinoma in rabbit by transarterial embolization-mediated siRNA delivery.

IntroductionHepatocellular carcinoma is currently the second leading cause of cancer-related deaths worldwide with an increasing incidence.ObjectiveThe objective of this study is to investigate the effect of vascular endothelial growth factor small interfering RNA (VEGF-siRNA) on rabbit VX2 carcinoma cell viability in vitro and the effect of transarterial embolization (TAE)-mediated VEGF-siRNA delivery on the growth of rabbit VX2 liver-transplanted model in vivo.MethodsQuantitative reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blot technologies were used to detect the expression level of VEGF. TAE and computed tomography scan were used to deliver the VEGF-siRNA and detect the tumor volume in vivo, respectively. Microvessel density was detected by immunohistochemistry with CD34 antibody. A biochemical autoanalyzer was used to evaluate the hepatic and renal toxicity.ResultsThe designed VEGF-siRNAs could effectively decrease the expression levels of VEGF mRNA and protein in vitro and in vivo. In vitro, the viability of rabbit VX2 carcinoma cells was reduced by 38.5%±7.3% (VEGF-siRNA no 1) and 30.0%±5.8% (VEGF-siRNA no 3) at 48 hours after transfection. Moreover, in rabbit VX2 liver-transplanted model, the growth ratios of tumors at 28 days after TAE-mediated siRNA delivery were 155.18%±19.42% in the control group, 79.67%±19.63% in the low-dose group, and 36.09%±15.73% in the high-dose group, with significant differences among these three groups. Microvessel density dropped to 34.22±4.01 and 22.63±4.07 in the low-dose group and high-dose group, respectively, compared with the control group (57.88±5.67), with significant differences among these three groups. Furthermore, inoculation of VX2 tumor into the liver itself at later stage induced significant increase in alanine aminotransferase and aspartate aminotransferase, indicating an obvious damage of liver functions, while treatment of VX2 tumor via TAE-mediated VEGF-siRNA had no toxicity to the livers and kidneys of rabbits, and VEGF-siRNA had the ability to protect liver damage induced by tumor growth.ConclusionThis is the first study to demonstrate that targeting VEGF via TAE-mediated siRNA delivery may become a powerful new option for effective treatment of hepatocellular carcinoma in the clinic.

The novel EZH2 inhibitor, GSK126, suppresses cell migration and angiogenesis via down-regulating VEGF-A.

To explore the effects and mechanisms of GSK126, a novel inhibitor of histone methyltransferase enhancer of zeste homologue 2, on cancer cell migration. Gastric cancer cell line MGC803 and human lung adenocarcinoma cell line A549 were treated with GSK126 at three doses. Transwell and wound healing assays were conducted to detect cell migration. Human umbilical vein endothelial cells tube formation assay and chick embryo chorioallantoic membrane assay were performed to assess the effects of GSK126 on angiogenesis in vitro and in vivo, respectively. The mRNA level of VEGF-A was detected by quantitative PCR, and the protein levels of VEGF-A were detected both by western blot analysis and immunohistochemistry. Epi-fluorescent intensity was obtained by in vivo imaging. GSK126 inhibited cell migration in both MGC803 and A549 in a dose-dependent manner, as revealed by transwell and wound healing assays. The effects of GSK 126 were similar to those of gefitinib at the same doses. Moreover, GSK126 at doses of 20 and 50µM inhibited angiogenesis both in vitro and in vivo. GSK126 reduced both the mRNA and protein expression of VEGF-A in a dose-dependent manner. Finally, in vivo imaging assay revealed that GSK126 at 200mg/kg significantly inhibited cancer cell migration. GSK126 inhibits cell migration and angiogenesis in solid tumor cell lines through down-regulation of VEGF-A expression. Thus, it may be considered as a novel anticancer drug candidate for solid tumor.

Estrogen deficiency in ovariectomized rats: can resistance training re-establish angiogenesis in visceral adipose tissue?

OBJECTIVE:The purpose of this study was to investigate the effects of resistance training on angiogenesis markers of visceral adipose tissue in ovariectomized rats.METHOD:Adult Sprague-Dawley female rats were divided into four groups (n=6 per group): sham-sedentary, ovariectomized sedentary, sham-resistance training and ovariectomized resistance training. The rats were allowed to climb a 1.1-m vertical ladder with weights attached to their tails and the weights were progressively increased. Sessions were performed three times per week for 10 weeks. Visceral adipose tissue angiogenesis and morphology were analyzed by histology. VEGF-A mRNA and protein levels were analyzed by real-time PCR and ELISA, respectively.RESULTS:Ovariectomy resulted in higher body mass (p=0.0003), adipocyte hypertrophy (p=0.0003), decreased VEGF-A mRNA (p=0.0004) and protein levels (p=0.0009), and decreased micro-vascular density (p=0.0181) in the visceral adipose tissue of the rats. Resistance training for 10 weeks was not able to attenuate the reduced angiogenesis in the visceral adipose tissue of the ovariectomized rats.CONCLUSION:Our findings indicate that the resistance training program used in this study could not ameliorate low angiogenesis in the visceral adipose tissue of ovariectomized rats.

Preventive Effects of the Chinese Herbal Medicine Prescription Tangkuei Decoction for Frigid Extremities on Sciatic Neuropathy in Streptozotocin-Induced Diabetic Rats.

Ischemia and hypoxia are important physiological changes in diabetic peripheral neuropathy (DPN). Chinese herbal medicine prescription Tangkuei Decoction for Frigid Extremities (TDFE) is useful for increasing blood flow. To help determine whether TDFE could protect the peripheral nerves of diabetic patients from the degeneration caused by high blood glucose, TDFE was administered to streptozotocin-induced diabetic rats for 6 or 12 weeks. Plantar thermal stimulation reaction time thresholds, sciatic nerve conduction velocities, and the levels of HIF-1α mRNA, HIF-1α protein, VEGF protein, and the endothelial marker vWF in sciatic nerves were measured at the end of the sixth and twelfth weeks. The thermal thresholds and sciatic nerve conduction velocities of the rats differed after 12 weeks, and the sciatic nerves of the diabetic rats that were given TDFE displayed higher levels of HIF-1α protein, VEGF protein, and HIF-1α mRNA than those of the diabetic model rats. The results at 6 weeks differed from those at 12 weeks. These results suggest that the early preventive application of TDFE effectively delayed the development of DPN and that TDFE increased HIF-1α mRNA levels in the sciatic nerves of diabetic rats through 12 weeks of treatment.

Increased MG-63s invasion potential mediated by HFs

During a malignant transformation, the crosstalk between the stroma and the cancer cells is described as a growing network of physical and paracrine signals, and it seems to have a direct influence on the phenotypic, genetic and epigenetic changes that affect the cells (1). In order to invade and metastasize to distant tissues, cancer cells transform themselves via ECM, induce tumor angiogenesis as well as undergo proliferation, detachment, migration, and invasion through secretion of various tumor derived factors (2). In this study we decided to analyze morphological and molecular aspects due to the coexistence between tumor cells MG-63s and fibroblasts HFs, verifying in particular the ability of MG-63s of invasion and microenvironment modulation. Monolayers of co-cultured cells were morphologically analyzed in time-laps by HR-SEM microscopy and a trans-well migration assay was performed over 24 h, 48 h, 72 h, and 96 h. The expression of several proteins, focusing on those involved in cancer cell invasion, inflammatory responses, and angiogenesis (TNF alpha, IL-6, YKL-40, MMP-1, MMP-9, and VEGF) was validated by Western blotting analysis. The images in time-laps for HR - SEM showed that fibroblasts in contact with MG-63 lost their spatial orientation, while the MG-63 quickly reached the confluence advancing towards HF cells, invading their space and overlying them. The increased MG-63s invasion mediated by the coexistence with HFs was confirmed by invasion assays in transwell co-culture. The protein levels of TNF-alpha, IL-6, YKL-40 and VEGF confirmed that tumor cells can regulate the development of a “tumor-stroma” via the aberrant expression of growth factors in the stromal compartment. Our results showed how tumor-stroma interactions play a significant role in tumor development and progression.

A promising scaffold with excellent cytocompatibility and pro-angiogenesis action for dental tissue engineering: Strontium-doped calcium polyphosphate.

Angiogenesis is of great importance in dental tissue engineering. Therefore, it is quite necessary for dental tissue engineering to develop a new scaffold with bioactivity to induce angiogenesis. This study was performed to determine the ability of 1% strontium-doped calcium polyphosphate (SCPP) to induce angiogenesis by detecting the levels of VEGF and bFGF from cultured human dental pulp cells (hDPCs). Results of scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) tests indicated the growth of hDPCs on 1%SCPP scaffolds was more superior than that on calcium polyphosphate (CPP) and hydroxyapatite (HA) scaffolds. The detection of MTT indicated 1%SCPP could accelerate proliferation of hDPCs. Further analyzed with ELISA demonstrated the protein levels of VEGF, bFGF from cultured hDPCs on 1%SCPP scaffolds were significantly higher than that on CPP and HA scaffolds. These findings above suggested 1%SCPP could be a promising scaffold for dental tissue engineering to induce angiogenesis.

Epidermal growth factor-like domain 7 promotes cell invasion and angiogenesis in pancreatic carcinoma.

Epidermal growth factor-like domain 7 (EGFL7), also known as vascular endothelial stain, was firstly identified as a modulator of smooth muscle cell migration. Though the expression of EGFL7 was reported to be up-regulated during tumorigenesis, the clinical and biological functions of EGFL7 in pancreatic carcinoma (PC) were still not fully elucidated. In this study, we found that the serum EGFL7 level in PC tissues was statistically higher than that in normal subjects (p<0.001), and its level in non-resectable patients was also higher than that in resectable ones (p=0.013). Among these resectable PC patients, the postoperative EGFL7 expression was significantly down-regulated when tumors were resected (p=0.018). Using the immunohistochemistry method, our results demonstrated that the positive expression of EGFL7 was significantly associated with the TNM stage (p=0.024), lymph node metastasis (p=0.003) and local invasion (p=0.022), and the EGFL7 expression closely correlated to the micro-vessel density (MVD) in PC tissues by Spearman analysis (r=0.941, p=0.000). In vitro, EGFL7 was silenced by the small interference RNA in PC cells, and our data indicated that down-regulation of EGFL7 did not influence the cycle progression, proliferation, colony formation and apoptosis of PC cells (p>0.05), whereas inhibition of EGFL7 expression could decrease PaCa-2 cell invasion (p<0.05). More interestingly, by tubular formation, Chick embryo chorioallantoic membrane (CAM) and ELISA assays, our results revealed that silencing EGFL7 expression represented a strong inhibiting effect on tubular formation of micro-vessels through down-regulating the protein levels of VEGF and Ang-2 (p<0.05). Our results raised the possibility of using EGFL7as a potential prognostic biomarker and therapy target of PC, and down-regulation of EGFL7 might be considered to be a potentially important molecular treatment strategy for patients with PC.

MiR-497 suppresses angiogenesis in breast carcinoma by targeting HIF-1α.

Angiogenesis is a key factor in the growth and dissemination of malignant diseases, including breast cancer, with significant implications for its clinical management. It is known that microRNAs (miRNAs) play important roles in regulating tumor properties in cancers. However, whether miR-497 contributes to breast cancer angiogenesis remains unknown. Our study demonstrated that miR-497 was significantly downregulated in breast cancer tissue samples and cell lines. Conditioned medium obtained from breast cancer cell line MCF-7, treated with miR-497 mimics, suppressed the proliferation and tube formation of human umbilical vein endothelial cells in vitro, in comparison with the untransfected cells or cells transfected with the control vector alone. Furthermore, western blot assay confirmed that the overexpression of miR-497 reduced VEGF and HIF-1α protein levels. In addition, stable transfection of miR-497 inhibited tumorigenicity and angiogenesis in vivo. Moreover, HIF-1α was also increased in the breast cancer cells under a hypoxic condition, while the ectopic expression of miR-497 partially restored its level. Taken together, our findings indicate that miR-497 is a potential target for the biological therapy of breast cancer. Moreover, miR-497 inhibited the growth of tumors and reduced angiogenesis in a nude mouse xenograft tumor model, which was probably caused by the downregulation of pro-angiogenic molecules, such as VEGF and HIF-1α.

Interleukin-8: A potent promoter of angiogenesis in gastric cancer.

Angiogenesis is a critical process in the development of tumor malignancy and occurs at various stages of tumor progression. Interleukin-8 (IL-8) is a pro-angiogenic factor produced by tumor-infiltrating macrophages that has been revealed to facilitate the development of angiogenesis in various cancers. However, whether IL-8 activates angiogenesis in gastric cancer remains unclear. The present study investigated the effect of IL-8 on the migration and canalization capacities of human umbilical vein endothelial cells (HUVECs). In addition, the protein and messenger RNA (mRNA) expression of selected angiogenesis markers, consisting of vascular endothelial growth factor (VEGF)-A, VEGF receptor (VEGFR)-1 and VEGFR-2, were assessed in the HUVECs. The HUVECs were co-cultured with human gastric cancer SGC7901 cells and exposed to various concentrations of IL-8 (0, 0.2, 0.5, 0.8 and 1.0 ng/ml). The migration and canalization abilities of the cells were detected by Transwell chamber and tube formation assays. Protein expression was detected using immunofluorescence and western blot analysis, and mRNA levels were assessed using reverse transcription quantitative polymerase chain reaction. The protein and mRNA levels of VEGF-A, VEGFR-1 and VEGFR-2 were measured in HUVECs cultured for 24 h. IL-8 at concentrations of 0.5, 0.8 and 1.0 ng/ml significantly promoted HUVEC cell migration (P=0.005, P=0.001 and P<0.001, respectively) and tube formation (P=0.039, P=0.003 and P<0.001, respectively). IL-8 at concentrations of 0.2, 0.5, 0.8 and 1.0 ng/ml significantly elevated the protein levels of VEGF-A (P<0.001) and VEGFR-2 (P=0.034, P<0.001, P<0.001 and P<0.001, respectively). IL-8 at concentrations of 0.8 and 1.0 ng/ml significantly elevated the protein levels of VEGF-1 (P=0.037 and P=0.002, respectively). Similarly, IL-8 at concentrations of 0.5, 0.8 and 1.0 ng/ml significantly upregulated the mRNA levels of VEGF-A (P=0.046, P=0.001 and P<0.001, respectively) and VEGFR-1 (P=0.042, P<0.001 and P<0.001, respectively). IL-8 at concentrations of 0.2, 0.5, 0.8 and 1.0 ng/ml significantly upregulated the mRNA levels of VEGFR-2 (P=0.003, P=0.005, P<0.001 and P<0.001, respectively). In conclusion, IL-8 may be a potent promoter of angiogenesis in gastric cancer.

Migration of Acute Lymphoblastic Leukemia Cells into the Central Nervous System Is Regulated By VEGF

In acute lymphoblastic leukemia (ALL), central nervous system (CNS) directed therapy is required to achieve long-term remission and survival even in patients without detectable CNS disease, indicating subclinical CNS manifestation in many patients. Hence, prophylactic CNS therapy is indispensable but bears the risk of secondary neoplasms and neurocognitive deficits in ALL survivors. Therefore, a better understanding of mechanisms mediating CNS involvement in ALL are needed in order to identify potential targets for prophylactic and therapeutic intervention.In this study, we transplanted primary patient B cell precursor (BCP) ALL cells onto NOD/SCID mice and investigated engraftment of human leukemia cells in the recipient's peripheral blood (PB), bone marrow (BM), spleen (S), and meninges by flowcytometry staining for huCD19. Upon disease onset, we identified meningeal infiltration of human ALL cells together with leukemia engraftment in BM, S and PB in a subset of samples (CNSpos) in contrast to absent CNS involvement despite high leukemia cell infiltration of BM, S and PB in other recipients (CNSneg). CNSpos and CNSneg phenotypes were consistently observed in subsequent xenograft passages. In CNSpos animals meningeal leukemia infiltration was also detected by immunohistochemistry on brain sections and meningeal enhancement was detected by small animal magnetic resonance imaging in CNSpos recipients.We further characterized ALL cells isolated from meningeal and BM infiltrates by gene expression profiling and identified the gene coding for vascular endothelial growth factor A (VEGF) to be highly expressed in ALL cells isolated from the CNS as compared to BM derived cells. Differential expression of VEGF was validated by qPCR and confirmed in independent sample cohorts. Most interestingly, reported functions of VEGF include regulation of cellular growth, vascular permeability, and trans-endothelial cell migration, and elevated VEGF protein levels have previously been described in cerebrospinal fluid specimens collected from ALL and AML patients with CNS leukemia.VEGF signals through its receptors 1 or 2 (VEGFR1/2). On all primary ALL samples, only expression of VEGFR1 but not VEGFR2 was detected. We used the BCP-ALL cell line Nalm-6, which also expresses VEGF and VEGFR1 and mediates a clear CNSpos phenotype upon engraftment. However, cellular survival, proliferation, apoptosis, and metabolic activity were not affected, neither upon exposure to VEGF or the antagonizing VEGF antibody bavacizumab, nor by stable VEGF overexpression or knock-down, thus indicating absence of autocrine VEGF/VEGFR1 signaling.We further analyzed whether paracrine VEGF signaling through VEGFR2, which upon activation mediates endothelial cell permeability, is involved in trans-endothelial migration of ALL cells leading to leukemia infiltration of the CNS. Brain endothelial cells (bEND.3) incubated with VEGF showed increased phosphorylation of VEGFR2 downstream signaling molecules (Src, AKT), which indicates activated signaling mediating cellular permeability.Moreover, we modeled migration of ALL cells through brain endothelial cells in a transwell assay and observed significantly increased migration of Nalm-6 ALL cells through bEND.3 monolayers upon exposure to VEGF or upon overexpression of VEGF as compared to controls. Vice versa, significantly lower numbers of migrated leukemia cells were detected after incubation with bevacizumab or upon VEGF knock down indicating VEGF dependent trans-endothelial migration.Thus, we identified high expression of VEGF in CNS derived leukemia cells, absent autocrine signaling of VEGF on ALL cells and, most importantly, VEGF dependent trans-endothelial migration of ALL cells indicating VEGF as a possible mediator of CNS leukemia.Finally, we investigated the impact of VEGF on CNS leukemia manifestation in vivo. Recipient animals transplanted with 3 CNSpos primograft samples (4 experiments, one in repetition) were treated with bevacizumab or control. In all experiments, anti-VEGF treatment significantly reduced the leukemia burden exclusively in the CNS but not in BM, S, and PB compartments, indicating that transmigration of leukemia cells and CNS manifestation in ALL is mediated by VEGF. Thus, targeting of VEGF signaling may serve as a novel strategy to control CNS leukemia in patients. DisclosuresNo relevant conflicts of interest to declare.